CN102070719A - Soluble leukemia stem cell targeting proteins TrxHis-hCD47 - Google Patents

Abstract

The invention relates to a soluble protein TrxHis-hCD47 targeted by leukemia stem cells, which is composed of the extracellular segment of human CD47 protein, namely amino acids 1-142, and a TrxHis expression tag, and the expression carrier is pET32a. After transforming Escherichia coli BL21, it was induced at 25°C for 24 hours to achieve soluble fusion expression with TrxHis, purified with a nickel metal chelate column for the His tag, and purified TrxHis-hCD47 protein was obtained after protein cleavage. TrxHis-hCD47 can be synthesized in large quantities through genetic engineering in vitro, and when applied in vivo, it can bind to the inhibitory receptor SIRP-α (signal regulatory protein alpha) on macrophages, preventing the combination of CD47 on leukemia stem cells and SIRP-α , so as to promote the phagocytosis of leukemia cells and leukemia stem cells by macrophages, which is of great significance for the treatment of leukemia.

Description

A leukemic stem cell targeting soluble protein TrxHis-hCD47

technical field

The invention relates to a soluble protein, which is a leukemic stem cell-targeting soluble protein TrxHis-hCD47 composed of the extracellular segment of human SIRPα ligand CD47 and a TrxHis expression tag fused at the amino terminal, and belongs to the technical field of medicine. the

Background technique

CD47, also known as integrin-associated protein (IAP), was initially recognized by co-purification from human placenta and integrin αVβ3 and co-immunoprecipitation from platelets and β3 integrin. Its function is related to integrin, so Call it IAP[1]. CD47 is a ubiquitously expressed antigen, present on different cells in all tissues. It is a 50kD membrane glycoprotein that belongs to the immunoglobulin (Ig) superfamily. Its extracellular segment, that is, the N-terminal, is an IgV-like domain, followed by a highly hydrophobic five-times transmembrane region, and the cytoplasmic region, that is, the C-terminal, is four different spliced bodies with a length of 3-36 amino acids. CD47 originally functioned as an integrin-dependent regulator of leukocyte responses to extracellular matrix proteins, but in addition, CD47 has many other important functions. The combination of CD47 on red blood cells and other normal cells in the body and SIRPα on macrophages can regulate the targeted clearance of macrophages, thereby preventing the phagocytosis of normal cells by macrophages. There is a relatively stable interaction between CD47 and αIIbβ or αVβ3 on platelets, αVβ3 smooth muscle cells on melanoma cells and ovarian tumor cells, and α2β1 on platelets, which promote platelet activation, aggregation, and chemotaxis of tumor cells and smooth muscle cells with diffusion. CD47 is also the receptor of extracellular matrix protein thrombospondin (thrombospondin, TSP). The combination of CD47 and monoclonal antibody and thrombospondin can induce caspase-dependent apoptosis. CD47 is a co-stimulator of T cell activation, activates the apoptotic process, and induces T cell anergy. As the extracellular ligand of SIRPα, CD47 plays an important role in the process of macrophages clearing tumor cells. the

Signal Regulatory Protein α (SIRPα) is a member of the immunoglobulin superfamily, with three immunoglobulin-like domains in the extracellular region and a tyrosine phosphorylation binding site in the cytoplasmic region. CD47 interacts with SIRP-a on macrophages, phosphorylates the ITIM motif on SIRPa, recruits protein tyrosine phosphatases containing SH2 domains to the membrane, and inhibits the aggregation of myosin on the cell surface, thereby Inhibits phagocytosis. Protein tyrosine phosphatases containing SH2 domains include SHP-1 and SHP-2, both of which are non-transmembrane protein tyrosine phosphatases. SHP-1 is mainly expressed on hematopoietic cells and reverse regulates many cellular functions; SHP-2 Widely expressed, regulates the function of small GTP-binding proteins Ras and Rho, and positively regulates cell function. The earliest evidence to determine the role of CD47 in regulating phagocytosis is that RBCs from CD47-deficient mice were quickly cleared after transplantation into wild mice, and CD47 on the surface of RBCs combined with SIRP-a on the surface of macrophages to transmit inhibitory signals. It conflicts with the phagocytic signal generated by the combination of macrophage surface receptors and phagocytic ligands, preventing red blood cells from being phagocytized. the

Studies have shown that for leukemia, the phagocytosis of macrophages is an important regulator of tumor immune surveillance. The up-regulation of CD47 expression on myeloid, lymphoid leukemia cells and leukemia stem cells can prevent leukemia by interacting with SIRPa. The cells are cleared by the host's innate immune system. This is also one of the important reasons for leukemia drug resistance and relapse. Blocking the interaction between CD47/SIRPα by monoclonal antibodies can promote the phagocytosis of leukemia cells and leukemia stem cells by macrophages, which has important therapeutic significance for the treatment of leukemia. the

Based on the important role of CD47 in regulating phagocytosis, the present invention expresses soluble CD47 extracellular segment protein, which competes with CD47 on leukemia cells to bind to SIRPα on macrophages, thereby promoting the phagocytosis of tumor cells by macrophages

Contents of the invention

The purpose of the present invention is to provide a leukemic stem cell targeting soluble protein TrxHis-hCD47, which can bind to the surface of macrophage cells and effectively enhance the phagocytosis of macrophages on leukemia cells and leukemia stem cells. the

The technical solution of the present invention is: a leukemia stem cell targeting soluble protein TrxHis-hCD47, characterized in that it is composed of the extracellular segment of human CD47 protein and a fused TrxHis expression tag; wherein the extracellular segment of hCD47 protein is hCD47 first -142 amino acids. the

The 1-142th amino acid of hCD47 contains an IgV-like domain responsible for binding to the SIRPα receptor; the fusion TrxHis expression tag is the amino terminal of the soluble protein; wherein, the amino acid sequence is No1, and is composed of No2 Nucleic acid sequence code:

The leukemia stem cell targeting soluble protein TrxHis-hCD47 has the amino acid sequence of No3 and is encoded by the nucleic acid sequence of No4:

The TrxHis expression tag fused at the amino terminal comprises the amino acid sequence of No5 and is encoded by the nucleic acid sequence of No6:

The leukemia stem cell-targeting soluble fusion protein TrxHis-hCD47 is solublely expressed in Escherichia coli BL21 by a low-temperature induction method, and purified with a nickel metal chelation column for the His tag, and pure TrxHis-hCD47 protein is obtained after protein cleavage. the

The present invention is characterized in that: TrxHis-hCD47 can be produced in large quantities through genetic engineering in vitro, and can be combined with inhibitory receptor SIRP-α (signal regulatory protein alpha) on macrophages when applied in vivo to prevent leukemia cells from CD47 on leukemia stem cells binds to SIRP-α, thereby promoting the phagocytosis of leukemia cells and leukemia stem cells by macrophages, which is of great significance for the treatment of leukemia. the

Description of drawings

Figure 1. The structure, construction and mode of action of hCD47 extracellular protein;

Fig. 2, the agarose gel electrophoresis of the PCR amplification product of target gene (hCD47);

Figure 3. Schematic diagram of the structure of the expression vector pET32a-hCD47;

Fig. 4. Enzyme digestion identification diagram of the expression vector pET32a-hCD47;

Fig. 5, the protein electrophoresis picture of TrxHis-hCD47 fusion protein expression;

Figure 6, electrophoresis of the purification of TrxHis-hCD47 fusion protein; Western blot qualitative detection:

Figure 7. Activity detection diagram of TrxHis-hCD47 fusion protein;

Figure 8. The number of macrophages engulfed in Jurkat cells was counted under a fluorescent microscope. the

Detailed ways

The present invention will be described in detail below with reference to the accompanying drawings and embodiments. the

1. Construct the expression vector. Figure 1 is a diagram of the structure, construction and mode of action of the fusion protein. Primers P1: 5'-GATATCATGTGGCCCCTGGTAGCGG-3' were designed according to the sequence of hCD47; P2: 5'-GGATCCATTTTCATTTGGAGAAAACC-3'. Using human lymph node cDNA as a template, polymerase chain reaction amplified the polynucleotide sequence encoding the amino acids of the extracellular segment of hCD47 (Figure 2), recovered by 1.5% agarose electrophoresis, ligated with pMD18-T vector at 16°C for 2 hours, and transformed by heat shock Escherichia coli XL10, after amplification, the target fragment was subcloned into the expression vector pET32a(+), pET32a-hCD47 was constructed (Figure 3), restricted enzyme digested (Figure 4), and sequenced for identification. the

Figure 1. Schematic diagram of the structure, construction and mode of action of hCD47 extracellular segment protein. the

Figure 2. Agarose gel electrophoresis of the PCR amplification product of the target gene (hCD47). Lane 1 (M) is the molecular weight marker (DL2000), and the arrow points to the amplified fragment. the

Fig. 3. Schematic diagram of the structure of the expression vector pET32a-hCD47. the

Fig. 4 is the enzyme digestion identification diagram of expression vector pET32a-hCD47; (plasmid is cut with restriction endonuclease EcoRV+BamHI double enzyme and EcoRI single enzyme respectively, agarose gel electrophoresis observation result. M track is marker DL2000, 1 Lanes -4 are the enzyme digestion results of different clones)

2. Induced expression of fusion protein. The expression vectors pET32a(+) and pET32a-hCD47 were respectively transformed into Escherichia coli BL21 by heat shock method, spread on LB plates containing 100 μg/ml ampicillin, cultured at 37°C for 12 hours, picked single clones and inoculated them on LB plates containing 100 μg/ml ampicillin LB liquid medium, 200rpm, cultured at 37°C for 12h, transferred to fresh LB(+) medium at 1%, 200rpm, cultured at 37°C for 3h, added IPTG with a final concentration of 1.0mM, 200rpm, and cultured at 25°C for 24h. the

3. Obtain the inclusion body protein of TrxHis-hCD47. Collect the bacteria by centrifugation, resuspend the bacteria in 200μl/ml culture medium PBS, lyse the bacteria by ultrasonication, add 1% Triton-X100, mix well, let stand at 4°C for 30 minutes, collect the supernatant and precipitate by centrifugation at 4°C, detect by SDS-PAGE, TrxHis - hCD47 protein mainly exists in the pellet in the form of inclusion body (Fig. 5). the

Fig. 5 is a protein electrophoresis image of TrxHis-hCD47 fusion protein expression. Transform the plasmids pET32a(+) and pET32a-hCD47 into Escherichia coli BL21 respectively. After small-volume culture, IPTG induces the expression of the fusion protein, and then take the whole bacterial lysate, the lysed supernatant (soluble fraction) and the lysed precipitate (ie, the inclusion body) SDS-PAGE analysis was performed separately. M is the molecular weight mark (from top to bottom are 170KD, 130KD, 100KD, 70KD, 55KD, 40KD, 35KD, 25KD, 15KD, 10KD)

4. Purification of inclusion body protein. pET32a(+) empty carrier protein TrxHis was purified from bacterial lysate supernatant, TrxHis-hCD47 was purified from inclusion body protein with nickel ion chelation column (Invitrogen ProBondTM), and all purification steps were performed according to ProBondTM purification manual. The purified protein was subjected to SDS-PAGE, and then Western blot was performed with an anti-His tag antibody, and a protein band of the correct size could be detected in TrxHis-hCD47 (Figure 6). the

Figure 6 shows the purification and Western blot detection of TrxHis-hCD47 fusion protein. From the renaturation supernatant of transformed Escherichia coli BL21 transformed with plasmid pET32a-hCD47, the protein containing His tag was purified with a nickel metal chelating column, and analyzed by SDS-PAGE. Track M is a protein molecular weight marker. The figure below is the result of Western blot detection of the fusion protein, and the primary antibody is an anti-His tag antibody. the

5. Determination of protein activity: the mouse macrophage cell line RAW264.7 was seeded in a 12-well plate at a density of 2×10 ⁵ /well, and adhered to the wall overnight. Then add Dio-labeled human T-lymphoblastic leukemia cell line Jurkat cells at a density of 2×10 ⁴ /well to the twelve-well plate pre-seeded with macrophages, and add CD47 extracellular domain protein (6 μg/ml) at the same time , Trx protein (6 μg/ml) was used as a control. Three replicate wells were set up for the treatment group and the control group respectively. After 2 hours of protein action, the number of macrophages that phagocytized Jurkat cells was counted under a fluorescence microscope, and dozens of fields of view were counted, and the results were statistically analyzed. (Figure 7, Figure 8)

FIG. 7 is a fluorescence microscope observation of the phagocytosis of Jurkat cells by macrophages in the control group and the treatment group. The arrows indicate the macrophages that phagocytized Jurkat cells. the

Figure 8 is the result of counting the number of macrophages swallowed into Jurkat cells under a fluorescence microscope, counting dozens of fields of view, and then statistically analyzing the data. Two-sample t-test, P<0.05, is statistically significant. the

The leukemic stem cell targeting soluble protein TrxHis-hCD47 of the present invention can be obtained from the above, which is composed of the extracellular segment of human CD47 protein and the fusion TrxHis expression tag; wherein the extracellular segment of hCD47 protein is amino acids 1-142 of hCD47 . the

The 1-142th amino acid of hCD47 contains an IgV-like structural domain responsible for binding to the SIRPα receptor; the fused TrxHis expression tag is the amino terminal of the soluble fusion protein. Wherein, the amino acid sequence is No1, and is encoded by the nucleic acid sequence of No2:

The leukemia stem cell targeting soluble fusion protein TrxHis-hCD47 has the amino acid sequence of No3 and is encoded by the nucleic acid sequence of No4:

The amino terminal fusion TrxHis expression tag includes the amino acid sequence of No5 and is encoded by the nucleic acid sequence of No6:

The leukemia stem cell-targeting soluble fusion protein TrxHis-hCD47 is solublely expressed in Escherichia coli BL21 by a low-temperature induction method, and purified with a nickel metal chelation column for the His tag, and pure TrxHis-hCD47 protein is obtained after protein cleavage. the

A leukemic stem cell-targeting soluble fusion protein TrxHis-hCD47 of the present invention can be mass-produced in vitro by genetic engineering, and can be combined with inhibitory receptor SIRP-α (signal regulatory protein alpha) on macrophages when applied in vivo, Preventing the combination of CD47 and SIRP-α on leukemia cells and leukemia stem cells, thereby promoting the phagocytosis of leukemia cells and leukemia stem cells by macrophages, is of great significance for the treatment of leukemia. the

sequence listing

the

<110> The Fourth Military Medical University of the Chinese People's Liberation Army

the

<120> A soluble protein TrxHis-hCD47 targeted by leukemia stem cells

the

<130> None

the

<160> 6

the

<170> PatentInversion3.3

the

<210> 1

<211> 331

No1: Amino acid sequence of TrxHis-hCD47 (No1 sequence referred to in the manual)

<212> PRT

<213> Artificial sequence

``

<400> 1

msdkiihltddsfdtdvlkadgailvdfwaewcgpckmiapildeiadeyqgkltvakln 60

the

idqnpgtapkygirgiptlllfkngevaatkvgalskgqlkefldanlagsgsghmhhhh 120

the

hhssglvprgsgmketaaakferqhmdspdlgtddddkamadimwplvaalllgsaccgs 180

the

aqllfnktksveftfcndtvvipcfvtnmeaqntevyvkwkfkgrdiytfdgalnkstv 240

the

ptdfssakievsqllkdaslkmdksdavshtgnytcevteltregetiielkyrvvswf 300

the

spnengsefelrrgacgrtrappppplrsgc 331

the

the

<210> 2

<211> 996

No2: Nucleic acid sequence of TrxHis-hCD47 (No2 sequence referred to in the instructions)

<212> DNA

<213> Artificial sequence

the

<400> 2

atgagcgataaaattattcacctgactgacgacagttttgacacggatgtactcaaagcg 60

the

gacggggcgatcctcgtcgatttctgggcagagtggtgcggtccgtgcaaaatgatcgcc 120

the

ccgattctggatgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaac 180

the

atcgatcaaaaccctggcactgcgccgaaatatggcatccgtggtatcccgactctgctg 240

the

ctgttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttg 300

the

aaagagttcctcgacgctaacctggccggttctggttctggccatatgcaccatcatcat 360

the

catcattcttctggtctggtgccacgcggttctggtatgaaagaaaccgctgctgctaaa 420

the

ttcgaacgccagcacatggacagcccagatctgggtaccgacgacgacgacaaggccatg 480

the

gctgatatcatgtggcccctggtagcggcgctgttgctgggctcggcgtgctgcggatca 540

the

gctcagctactatttaataaaacaaaatctgtagaattcacgttttgtaatgacactgtc 600

the

gtcattccatgctttgttactaatatggaggcacaaaacactactgaagtatacgtaaag 660

the

tggaaatttaaaggaagagatatttacacctttgatggagctctaaacaagtccactgtc 720

the

cccactgactttagtagtgcaaaaattgaagtctcacaattactaaaaggagatgcctct 780

the

ttgaagatggataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaaca 840

the

gaattaaccagagaaggtgaaacgatcatcgagctaaaatatcgtgttgtttcatggttt 900

the

tctccaaatgaaaatggatccgaattcgagctccgtcgacaagcttgcggccgcactcga 960

the

gcaccaccaccaccaccactgagatccggctgctaa 996

the

the

<210> 3

<211> 142

No3: The amino acid sequence of the extracellular segment of hCD47 (the No3 sequence referred to in the instructions)

<212> PRT

<213> Homosapiens

the

<400> 3

mwplvaalllgsaccgsaqllfnktksveftfcndtvvipcfvtnmeaqnttevyvkwkf 60

the

kgrdiytfdgalnkstvptdfssakievsqllkdaslkmdksdavshtgnytcevtelt 120

the

regetiielkyrvvswfspnen 142

the

the

<210> 4

<211> 426

  No4: The nucleic acid sequence of the extracellular segment of hCD47 (the No4 sequence referred to in the instructions)

<212> DNA

<213> Homosapiens

the

<400> 4

atgtggcccctggtagcggcgctgttgctgggctcggcgtgctgcggatcagctcagcta 60

the

ctatttaataaaacaaaatctgtagaattcacgttttgtaatgacactgtcgtcattcca 120

the

tgctttgttactaatatggaggcacaaaacactactgaagtatacgtaaagtggaaattt 180

the

aaaggaagagatattttacacctttgatggagctctaaacaagtccactgtccccactgac 240

the

tttagtagtgcaaaaattgaagtctcacaattactaaaaggatgcctctttgaagatg 300

the

gataagagtgatgctgtctcacacacaggaaactacacttgtgaagtaacagaattaacc 360

the

agagaaggtgaaacgatcatcgagctaaaatatcgtgttgtttcatggttttctccaaat 420

the

gaaaat 426

the

the

<210> 5

<211> 163

No5: Amino acid sequence of TrxHis (No5 sequence referred to in the instructions)

<212> PRT

<213> Artificial sequence

the

<400> 5

msdkiihltddsfdtdvlkadgailvdfwaewcgpckmiapildeiadeyqgkltvakln 60

the

idqnpgtapkygirgiptlllfkngevaatkvgalskgqlkefldanlagsgsghmhhhh 120

the

hhssglvprgsgmketaaakferqhmdspdlgtddddkamadi 163

       

the

the

<210> 6

<211> 489

No6: Nucleic acid sequence of TrxHis (No6 sequence referred to in the manual)

<212> DNA

<213> Artificial sequence

the

<400> 6

atgagcgataaaattattcacctgactgacgacagttttgacacggatgtactcaaagcg 60

the

gacggggcgatcctcgtcgatttctgggcagagtggtgcggtccgtgcaaaatgatcgcc 120

the

ccgattctggatgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaac 180

the

atcgatcaaaaccctggcactgcgccgaaatatggcatccgtggtatcccgactctgctg 240

the

ctgttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttg 300

the

aaagagttcctcgacgctaacctggccggttctggttctggccatatgcaccatcatcat 360

the

catcattcttctggtctggtgccacgcggttctggtatgaaagaaaccgctgctgctaaa 420

the

ttcgaacgccagcacatggacagcccagatctgggtaccgacgacgacgacaaggccatg 480

the

gctgatatc 489

Claims (5)

1. the soluble proteins TrxHis-hCD47 of a leukemic stem cells target is characterized in that: it is expressed label by the TrxHis of people CD47 extracellular fragment and fusion and forms; Wherein the CD47 extracellular fragment is meant CD47 the 1st～142 amino acids.

2. the soluble proteins TrxHis-hCD47 of a kind of leukemic stem cells target according to claim 1 is characterized in that: described hCD47 the 1st～142 amino acids comprises and SIRP α bonded IgV spline structure territory and upstream sequence; It is the N-terminal of this soluble proteins that the TrxHis of described fusion expresses label, and wherein, aminoacid sequence is No1, and by the nucleic acid sequence encoding of No2:

The aminoacid sequence of No1:TrxHis-hCD47

msdkiihltddsfdtdvlkadgailvdfwaewcgpckmiapildeiadeyqgkltvaklnidqnpgtapkygirgipt

lllfkngevaatkvgalskgqlkefldanlagsgsghmhhhhhhssglvprgsgmketaaakferqhmdspdlgtd

dddkamadimwplvaalllgsaccgsaqllfnktksveftfcndtvvipcfvtnmeaqnttevyvkwkfkgrdiytf

dgalnkstvptdfssakievsqllkgdaslkmdksdavshtgnytcevteltregetiielkyrvvswfspnengsefel

rrgacgrtrappppplrsgc

The nucleotide sequence of No2:TrxHis-hCD47

atgagcgataaaattattcacctgactgacgacagttttgacacggatgtactcaaagcggacggggcgatcctcgtcgatt

tctgggcagagtggtgcggtccgtgcaaaatgatcgccccgattctggatgaaatcgctgacgaatatcagggcaaactg

accgttgcaaaactgaacatcgatcaaaaccctggcactgcgccgaaatatggcatccgtggtatcccgactctgctgctg

ttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttgaaagagttcctcgacgctaacct

ggccggttctggttctggccatatgcaccatcatcatcatcattcttctggtctggtgccacgcggttctggtatgaaagaaa

ccgctgctgctaaattcgaacgccagcacatggacagcccagatctgggtaccgacgacgacgacaaggccatggctg

atatcatgtggcccctggtagcggcgctgttgctgggctcggcgtgctgcggatcagctcagctactatttaataaaacaaa

atctgtagaattcacgttttgtaatgacactgtcgtcattccatgctttgttactaatatggaggcacaaaacactactgaagtat

acgtaaagtggaaatttaaaggaagagatatttacacctttgatggagctctaaacaagtccactgtccccactgactttagt

agtgcaaaaattgaagtctcacaattactaaaaggagatgcctctttgaagatggataagagtgatgctgtctcacacacag

gaaactacacttgtgaagtaacagaattaaccagagaaggtgaaacgatcatcgagctaaaatatcgtgttgtttcatggttt

tctccaaatgaaaatggatccgaattcgagctccgtcgacaagcttgcggccgcactcgagcaccaccaccaccaccact

gagatccggctgctaa。

3. a kind of leukemic stem cells targeting soluble albumen TrxHis-hCD47 according to claim 1, it is characterized in that: described leukemic stem cells targeting soluble albumen TrxHis-hCD47 has the aminoacid sequence of No3, and by the nucleic acid sequence encoding of No4:

The aminoacid sequence of No3:hCD47 extracellular fragment

mwplvaalllgsaccgsaqllfnktksveftfcndtvvipcfvtnmeaqnttevyvkwkfkgrdiytfdgalnkstvpt

dfssakievsqllkgdaslkmdksdavshtgnytcevteltregetiielkyrvvswfspnen

The nucleotide sequence of No4:hCD47 extracellular fragment

atgtggcccctggtagcggcgctgttgctgggctcggcgtgctgcggatcagctcagctactatttaataaaacaaaatctg

tagaattcacgttttgtaatgacactgtcgtcattccatgctttgttactaatatggaggcacaaaacactactgaagtatacgt

aaagtggaaatttaaaggaagagatatttacacctttgatggagctctaaacaagtccactgtccccactgactttagtagtg

caaaaattgaagtctcacaattactaaaaggagatgcctctttgaagatggataagagtgatgctgtctcacacacaggaaa

ctacacttgtgaagtaacagaattaaccagagaaggtgaaacgatcatcgagctaaaatatcgtgttgtttcatggttttctcc

aaatgaaaat。

4. a kind of leukemic stem cells targeting soluble albumen TrxHis-hCD47 according to claim 2 is characterized in that: described N-terminal merges TrxHis expresses label, comprises the aminoacid sequence of No5, and by the nucleic acid sequence encoding of No6:

The aminoacid sequence of No5:TrxHis

msdkiihltddsfdtdvlkadgailvdfwaewcgpckmiapildeiadeyqgkltvaklnidqnpgtapkygir

giptlllfkngevaatkvgalskgqlkefldanlagsgsghmhhhhhhssglvprgsgmketaaakferqhmds

pdlgtddddkamadi

The nucleotide sequence of No6:TrxHis

atgagcgataaaattattcacctgactgacgacagttttgacacggatgtactcaaagcggacggggcgatcctcgtcgatt

tctgggcagagtggtgcggtccgtgcaaaatgatcgccccgattctggatgaaatcgctgacgaatatcagggcaaactg

accgttgcaaaactgaacatcgatcaaaaccctggcactgcgccgaaatatggcatccgtggtatcccgactctgctgctg

ttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttgaaagagttcctcgacgctaacct

ggccggttctggttctggccatatgcaccatcatcatcatcattcttctggtctggtgccacgcggttctggtatgaaagaaa

ccgctgctgctaaattcgaacgccagcacatggacagcccagatctgggtaccgacgacgacgacaaggccatggctg

atatc。

5. the soluble proteins TrxHis-hCD47 of a kind of leukemic stem cells target according to claim 1, it is characterized in that: described leukemic stem cells targeting soluble albumen TrxHis-hCD47 realizes solubility expression with the low temperature induction method in e. coli bl21, and use nickel metal-chelating column purification at the His label, obtain pure TrxHis-hCD47 albumen behind the protein cleavage.

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